PROJECT SUMMARY/ABSTRACT Heme is an ancient biological cofactor that plays critical roles in mitochondrial redox chemistry, energy production, and signaling in all eukaryotic cells from humans to microbial pathogens. Despite functional similarities in their reliance on heme, divergent eukaryotes have evolved distinct molecular adaptations that tune heme metabolism to particular cellular demands. Comparing and contrasting key features of mitochondrial heme metabolism can thus reveal general principles in heme trafficking and utilization, as well as unveil unique adaptations that highlight the evolutionary diversity among eukaryotes. Elucidating these general principles will increase understanding of heme-related mitochondrial dysfunctions that underpin major human diseases. Understanding of functional differences in heme metabolism can be exploited to develop new pathogen-specific therapies that avoid human toxicity. In this proposal, we lay out a long-term goal of our research program to deeply understand key biochemical properties and mechanisms of mitochondrial heme metabolism in humans and a major human pathogen. In pursuing this goal over the next five years, we will focus our studies on understanding the molecular mechanisms of biogenesis and function of cytochrome c, a critical and tractable heme-binding protein with major unresolved mechanistic features that exemplifies key features of mitochondrial heme metabolism and its conservation as well as diversity between eukaryotes. Cytochrome c has a conserved essential function in the mitochondrial electron transport chain (ETC) but also plays an important, separate role as a peroxidase that signals the onset of apoptosis. The molecular features of cytochrome c that underpin its functional transition from ETC to peroxidase activity remain sparsely defined. Cytochrome c is distinguished from other heme-binding proteins by its use of a conserved CXXCH sequence motif to covalently bind heme via stable thioether bonds. This covalent attachment requires the mitochondrial intermembrane space (IMS) enzyme, holo cytochrome c synthase (HCCS). Although HCCS function is fundamental to all eukaryotic respiration, the structure of HCCS is unknown and thus molecular insight into its mechanism of attaching heme to cytochrome c is lacking. Furthermore, it remains unknown how heme is trafficked to HCCS in the IMS from its site of synthesis within the mitochondrial matrix. We propose comparative studies between human cells and Plasmodium parasites that invade heme-rich human erythrocytes to tackle the following key challenges: 1) to define the molecular features of cytochrome c that mediate its transition between ETC and peroxidase activities; 2) to elucidate the three-dimensional structure of HCCS and unravel its molecular mechanism of heme attachment to cytochrome c; and 3) to determine the mechanism of heme acquisition by HCCS. This work will advance fundamental understanding of mitochondrial heme metabolism in humans and a critical human pathogen and provide a platform for longer-term integrative studies of heme metabolism that will test and extend the lessons learned from our initial focus on cytochrome c.